REVIEWS

Bioengineering for Salinity Tolerance in Plants: State of the Art

Pradeep K. Agarwal • Pushp Sheel Shukla •

Kapil Gupta • Bhavanath Jha

Published online: 27 April 2012

� Springer Science+Business Media, LLC 2012

Abstract Genetic engineering of plants for abiotic stress

tolerance is a challenging task because of its multifarious

nature. Comprehensive studies for developing abiotic stress

tolerance are in progress, involving genes from different

pathways including osmolyte synthesis, ion homeostasis,

antioxidative pathways, and regulatory genes. In the last

decade, several attempts have been made to substantiate

the role of ‘‘single-function’’ gene(s) as well as transcrip-

tion factor(s) for abiotic stress tolerance. Since, the abiotic

stress tolerance is multigenic in nature, therefore, the recent

trend is shifting towards genetic transformation of multiple

genes or transcription factors. A large number of crop

plants are being engineered by abiotic stress tolerant genes

and have shown the stress tolerance mostly at laboratory

level. This review presents a mechanistic view of different

pathways and emphasizes the function of different genes in

conferring salt tolerance by genetic engineering approach.

It also highlights the details of successes achieved in

developing salt tolerance in plants thus far.

Keywords Gene function � Genetic engineering � Salt

tolerance

Abbreviations

CBL Calcineurin B-like protein

CIPK CBL-interacting protein kinases

NPK1 Mitogen-activated protein kinase kinase kinase

NDPK2 Nucleoside diphosphate kinase 2

SAPK4 Sucrose nonfermenting 1-related protein

kinase2 (SnRK2)

AtMEK1 MAPK kinase

MYB Myeloblastoma

NAC No apical meristem, ATAF 1,2 and cup-shaped

cotyledon

DRE Drought responsive element

DBF DRE binding factor

DREB Drought responsive element binding protein

TPS Trehalose-6-phosphate synthase

p5cs D1-Pyroline-5-carboxylate synthase

codA Choline oxidase

BADH Betaine aldehyde dehydrogenase

mt1D Mannitol-1-phosphate dehydrogenase

P5CR P5C reductase

GutD Glucitol-6-phosphate dehydrogenase

MIPS L-Myo-Inositol-1-phosphate synthase

APX Cytosolic ascorbate peroxidase

DHAR Dehydroascorbate reductase

MDHAR Mono DHAR

SOD Superoxide dismutase

ADC Arginine decarboxylase

ODC Ornithine decarboxylase

SAMDC S-Adenosyl methionine decaroboxylase

SPDS Spermidine Synthase

NHX-1 Vacuolar Na?/H? antiporter

SOS1 Salt overly sensitive

TsVP H?-pyrophosphatase

HKT2 High efficiency potassium transport

Introduction

Plants being sessile are highly affected by harsh climatic

changes. Although plants gradually evolved a remarkable

P. K. Agarwal (&) � P. S. Shukla � K. Gupta � B. Jha

Discipline of Marine Biotechnology and Ecology, Central Salt

and Marine Chemicals Research Institute (Council of Scientific

and Industrial Research), G.B. Road, Bhavnagar 364021,

Gujarat, India

e-mail: pagarwal@csmcri.org

123

Mol Biotechnol (2013) 54:102–123

DOI 10.1007/s12033-012-9538-3

ability to adapt themselves to such a highly variable

environmental onslaughts, environmental stresses never-

theless cause over 50% crop loss [1]. Approximately, 7%

of total land area (1,000 million ha) and 20% of the irri-

gated agriculture land is affected by soil salinity [2] and

imposes major constraints to the sustainability of crop

yield.

Salinity imparts both ionic and osmotic stresses thus

limiting plant growth and productivity. Plants respond and

adapt to these conditions by regulating a wide array of

genes. Salinity causes adverse impact on plant growth by

disturbing the ionic equilibrium and eliciting sodium tox-

icity. High Na? concentration is toxic to cell metabolism

and has deleterious effects on the functioning of some of

the enzymes [3]. Different plants employ different mech-

anisms to minimize the damage from Na?, e.g., minimize

initial influx, maximize efflux, minimize loading into the

xylem, and maximize recirculation out of the shoot to

phloem, intercellular compartmentalization and even

secretion of salt from the leaf surface [4, 5].

Various genes induced by salt stress could be grouped

under two categories, namely, ‘‘single-function’’ genes and

regulatory genes. The first category of genes generally

facilitates production of protective metabolites, which

include osmolytes, transporters/channel proteins, antioxi-

dative enzymes, lipid biosynthesis genes, polyamines, etc.

The second class of genes consists of regulatory proteins

like bZIP, DREB, MYC/MYB, and NAC, which control

the expression of many downstream salt stress tolerant

genes [6, 7]. These genes converge and interact in different

pathways related to the abiotic stress and successfully lead

to tolerance (Fig. 1).

Different genes related to abiotic stress tolerance, have

been introduced into a variety of plants [1, 8–10]. Many

reviews have been published recently on oxidative pathway

[11], transporters [12–15] and role of regulators in con-

trolling salt stress tolerance [6, 7, 16–18]. The present

article focuses on new paradigm developed in the recent

past years in developing salt tolerant plants by employing

genes from all the possible pathways.

Signaling Molecules

Environmental signals are first perceived by signaling

molecules and involves protein phosphorylation, dephos-

phorylation, phospholipid metabolism, Ca2? sensing, etc.

Their overexpression in many plants showed better stress

tolerance (Table 1). Stress and other extracellular stimuli

change the intracellular Ca2? concentration [19, 20]. Ca2?

signaling process is considered as one of the earliest events

in salt signaling, and play an essential role in the ion

homeostasis leading to salt tolerance in plants [15, 21].

Calcineurin B-like proteins (CBL) sense the calcium signal

and participate in salt stress signal transduction pathway

and control the influx and efflux of Na?. The signaling

approach to improve salt tolerance was demonstrated in

transgenic tobacco by modulating the expression of a cal-

cium stress-signaling component from yeast, Ca2?/cal-

modulin dependent protein phosphatase [22]. The

transgenic tobacco plants co-expressing the two catalytic

and regulatory subunits of this protein exhibited substantial

NaCl tolerance [22]. The calcineurin B-like (regulatory)

Ca2? sensor, SOS3 (Salt overly sensitive 3) has been cloned

from Arabidopsis [23]. This protein triggers the activity of

SOS pathway for plant Na? tolerance, the SOS gene and

its applications are discussed below along with the

transporter genes. A CBL protein from maize, ZmCBL4,

showed salt stress tolerance in Arabidopsis. Expression of

35S:ZmCBL4 complemented the salt hypersensitivity in

Arabidopsis sos3 mutant and enhanced the salt tolerance

in wild-type Arabidopsis at the germination and seedling

stages [24]. Recently, Tripathi et al. [25] showed that

overexpression of a constitutively active mutant of Ca-

CIPK6 from chickpea promoted salt tolerance in trans-

genic tobacco. AtNDPK2 activated both AtMPK3 and

AtMPK6, and its overexpression showed enhanced salt

and other abiotic stress tolerance by reducing the ROS

concentration in the plants [26]. SAPK4 is a serine thre-

onine type of kinase and is known to regulate the stress

responsive gene expression. The overexpression of SAPK4

resulted in improved germination, growth and develop-

ment under salt stress. In response to salt stress, the

SAPK4-overexpressing rice accumulated less Na? and Cl-

and showed improved photosynthesis [27].

Plants use another common mechanism to translate

external stimuli into cellular responses through the acti-

vation of mitogen-activated protein (MAP) kinase cascade

[28]. MAPKs are signaling modules that phosphorylate

specific serine/threonine residues on the target protein

substrate and regulate a variety of cellular activities. The

MAPK cascade consists of three functionally interlinked

protein kinases: MAPKKK, MAPKK, and MAPK. Based

on sequence alignment, the MAPKKs are placed in four

groups (A–D). The roles of groups A, B, and C have been

studied in relation to biotic and abiotic stresses [29].

Overexpression of MAP kinases showed enhanced toler-

ance to salt stress (Table 1). Transgenic rice plants with

OsMAPK5 and OsMAPK44 genes showed tolerance to salt,

drought and cold stresses [30, 31]. The overexpression of

ZMKK4 from Zea mays in Arabidopsis plants showed

enhanced salt tolerance and also higher proline and soluble

sugar contents, and higher POD and CAT activities com-

pare to control plants. The transgenic plant showed

upregulation of the transcription factor, TF, which even-

tually control the expression of COR47, Rd29a, and P5CS

Mol Biotechnol (2013) 54:102–123 103

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[32]. Several researchers investigated that on one hand

MAP kinase control the stress in plants by overexpressing

of antioxidative genes and on other hand it activated the

TFs which further control the downstream genes. Kong

et al. [32] has studied that the transgenic plants showed

upregulation of CBF1, CBF2, CBF3, STZ, DREB2A TFs

and which in turn showed the enhanced expression of the

COR47, RD29A, P5CS2 genes. The signaling molecules

are reported to interact for developing cross tolerance, in

this case when one type of stress renders to plants than it

leads to resistant to another type of stress [33]. LeCDPK1

gene from tomato also is a good example of imparting

cross-tolerance, it interrelates the signaling responses to

wounding and salt stress. It showed that mechanical

wounding increases salt-stress tolerance by involving the

signaling peptide systemin and the synthesis of JA (Jas-

monic Acid) [34]. It is evident from the literature that

signaling genes play important role in abiotic stress toler-

ance by regulating the expression of salt tolerant genes and

TFs. In spite of the important role of signaling molecule,

only few studies had been carried out (Table 1) for engi-

neering salt tolerance in the plants, therefore it is desirable

to have more transgenic lines with this kind of genes in

future.

AtHK1, Ca++, SOS3, CDPKs,

SnRK1

AREB/ABF,bZIP, CBF/DREB, MYC/MYB, NAC, WRKY

Detoxification

Aquaporin and transporters HSps, LEAs, proteinases

Photosynthesis and related metabolism

Chaperone

Osmoprotection

Stress tolerance and resistance

Signal sensing

Transcription factor

Gene activation &Stress response

Fig. 1 A generic pathway under salt and drought stress. The signal is perceived by the sensor proteins, which in turn activates the transcription

factors (TFs). The TFs trigger the process of activating the genes of different class resulting to achieve stress tolerance

104 Mol Biotechnol (2013) 54:102–123

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Regulatory Genes

TFs interact with different cis-elements in the promoter

regions of various downstream genes and modulate their

expression. TFs play a pivotal role in developing stress

tolerance in plants against various environmental stresses

(Fig. 2). TFs corresponds to a large number in plants,

Arabidopsis genome contains 5.9% TFs [35]. TFs are better

expanded in plants due to significant complexity of plant

metabolism, as compared to other kingdoms [36]. TFs can

be classified into [50 different families, based on the

presence of their DNA-binding domains [35]. In Arabid-

opsis, cis-elements and corresponding binding proteins,

with distinct DNA-binding domains, such as AP2/ERF

(Apetala 2/Ethylene responsive factor), basic leucine zip-

per, HD-ZIP (homeodomain leucine zipper), MYC, MYB

(myelocytomatosis, myeloblastosis) and different classes

of zinc finger domains, have been identified [37]. The

control of specific sets of genes can be accomplished by the

combinatorial interaction among TFs, between TFs and

non-DNA-binding proteins, and between TFs and cis-reg-

ulatory elements [38]. Therefore, it is inevitable to study

the convergence and interaction horizontally or vertically

of TFs for better control of abiotic stresses.

ABA signaling is an important component of abiotic

stress transduction pathways. Besides ABA, salicylic acid-

mediated pathway also regulates TFs in abiotic stress. The

role of ABA in stress signaling and its involvement in

different regulatory systems during abiotic stress in ABA-

dependent and independent manner has been discussed by

Agarwal et al. [16] and Agarwal and Jha [6]. In this review,

we have incorporated only the recent developments in the

TFs and their role in only salt and drought tolerance in

plants.

DREBs

The ABA-independent stress-responsive genes are regulated

by dehydration-responsive element binding (DREB) pro-

teins, which bind to dehydration-responsive element (DRE)

cis-elements. DREBs are important plant-specific TFs,

which induce a set of abiotic stress related genes and impart

stress tolerance. DREB genes have been isolated and char-

acterized from a wide variety of plants, and their differential

regulation and functional analysis is reviewed by Agarwal

et al. [16] and Lata and Prasad [39]. A number of down-

stream genes get activated by overexpression of DREB TFs

leading to enhanced abiotic stress tolerance. Microarray

analysis of DREB transgenics showed higher expression of

large number of downstream genes belonging to late

embryogenic abundant (LEA) protein, heat shock, detoxifi-

cation, seed proteins and enzymes involved in metabolism,

etc. [6]. Recently, it has been reported that DREB2A from

Salicornia brachiata showed enhanced salt tolerance in E.

coli, indicating that SbDREB2A is interacting with tran-

scriptional network in the bacterial cells [40].

Table 1 Overexpression of signaling molecule to develop salt and desiccation tolerance

Gene/source Transgenic plant Performance of transgenic plant References

CBL4/maize Arabidopsis Salt tolerance [24]

CBL5/Arabidopsis Arabidopsis Salt and dehydration tolerance [165]

CIPK6/chickpea Tobacco Salt tolerance [25]

MAPK5/rice Rice Enhanced tolerance to drought, salt and cold stresses [30]

MAPK44/rice Rice Salt tolerance [31]

MEK1/Arabidopsis Arabidopsis Salt and dehydration tolerance [166]

MAPK kinase kinase (MAPKKK/DSM1) Rice Dehydration tolerance [167]

ZmMKK4/maize Arabidopsis Salt tolerance [32]

CIPK03, OsCIPK12, and CIPK15 Rice Salt, dehydration and cold tolerance [168]

AtCPK6/Arabidopsis Arabidopsis Salt and drought tolerance [169, 170]

NPK1 Maize Salt, dehydration, cold and heat tolerance [171]

NDPK2/Arabidopsis Arabidopsis Salt, cold and oxidative stress tolerance [26]

SAPK4/rice Rice Salt stress tolerance [27]

ZmSAPK8/maize Arabidopsis Salt tolerance [172]

GhMPK2/Gossypium hirsutum Tobacco Salt and drought tolerance [173]

OsMSR2/Oryza sativa Arabidopsis Salt and drought tolerance [174]

TaSnRK2.8/Triticum aestivum Arabidopsis Salt and drought tolerance [175]

CBL Calcineurin B-like protein, CIPK CBL-interacting protein kinases, NPK1 Mitogen-activated protein kinase kinase kinase, NDPK2Nucleoside diphosphate kinase 2, SAPK4 Sucrose nonfermenting 1-related protein kinase2 (SnRK2), AtMEK1 MAPK kinase

Mol Biotechnol (2013) 54:102–123 105

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NAC

Another plant-specific TF, NAC (NAM-ATAF1,2-CUC2),

regulates both the ABA-dependent and independent genes.

These TFs are expressed in different tissues at various

developmental stages and are involved in plant growth and

development [41]. The N-terminal region of the NAC

protein contains a highly conserved domain found in NAM

(no apical meristem), ATAF1, 2 and CUC2 (cup-shaped

cotyledon) proteins. The domain might form a helix-turn-

helix structure that specifically binds to target DNA [42].

The C-terminal region of NAC proteins possesses highly

divergent sequence. The first NAC gene isolated was NAM

from petunia [43], which played a critical role in deter-

mining shoot apical meristem and primordia positions [44].

Recently, NAC genes are also found to be involved in

abiotic and biotic stresses [45–47]. Arabidopsis NAC

genes, namely ANAC019, ANAC055, and RD26/ANAC072,

showed the upregulation of several stress-related genes and

conferred enhanced drought tolerance [48]. Later, Hu et al.

[49] reported that rice SNAC1 (Stress-ResponsiveNAC 1)

showed significantly better drought and salinity stress tol-

erance in transgenic rice. The over expression of this gene

resulted significantly increased stomata closure under

drought stress. Similarly, Hu et al. [50] reported that

SNAC2 (Stress-Responsive NAC 2) (identical to OsNAC6)

showed improved tolerance to various stresses in the

transgenic rice. The transgenic lines showed higher seed

germination on 150 mM NaCl compared to control plants

whereas there was no difference in the germination rate on

MS basal medium. The SNAC2 profiling analysis of

transgenic plants revealed many upregulated genes related

to stress response and adaptation such as peroxidase,

ornithine aminotransferase, heavy metal-associated protein,

sodium/hydrogen exchanger, heat shock protein, GDSL-

like lipase, and phenylalanine ammonia lyase. The up- or

downregulated genes compared in the SNAC1 and SNAC2

overexpression plants showed sharp difference. This may

be because of the difference in the conserved flanking sites

of the target genes between SNAC1 and SNAC2.

Another rice NAC gene, OsNAC6, was induced both by

biotic and abiotic stress. The transgenic lines showed

upregulation of many biotic and abiotic stress responsive

genes. The transgenic plants showed higher tolerance to

dehydration and salt stress. The OsNAC6 transgenic lines

with stress inducible promoters OSNAC6 and LIP9 (low-

Drought, Salt

A B A d e p e n d e n t A B A i n d e p e n d e n t

GENE EXPRESSION

PHYSIOLOGICAL AND BIOLOGICAL CHANGES

STRESS TOLERANCE

S I G N A L P E R C E P T I O N

MYC/ MYB

CBF4

DREB2AREB/ABF

NAC NAC

DRE/CRT (rd29A)

MYCRS, MYBRS (rd22)

NACRSNACRS(erd1)

ABRE(rd29B)

Fig. 2 A diagrammatic representation of ABA dependent as well as independent regulatory cellular signal transduction pathways between stress

signal perception and gene expression involved in stress responsive gene expression

106 Mol Biotechnol (2013) 54:102–123

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temperature-induced protein) minimized the negative

effect on growth compared to control plants and showed

higher tolerance in the presence of salt [47]. ONAC063

from rice showed enhanced tolerance to salt and osmotic

stress in Arabidopsis plants. Microarray analysis showed

upregulation of 29 genes among them oleosin and amylase

expressed more than 100-fold [51]. Two NAC genes

GmNAC11 and GmNAC20 have been isolated from the

soybean. These both genes differentially induced under

stress condition and plant hormones. The overexpression of

the GmNAC20 showed the tolerance to salinity and freez-

ing, whereas, GmNAM11 showed tolerance to only salinity.

Both genes likely regulated the stress tolerance by DREB/

CBF-COR pathway [52]. Liu et al. [53] reported that

overexpression of Chrysanthemum DgNAC1 in tobacco

resulted salt tolerance. OsNAC5 [54] when overexpressed

showed better salt tolerance than RNAi knock-out lines or

wt plants. Different physiological parameters of OsNAC5

overexpression lines showed that proline and free sugar

level get enhanced in transgenic lines. Further, it is also

observed that MDA and H2O2 content have got reduced in

overexpressed lines compared to knockdown lines [54].

Similarly, Takahashi et al. [55] also observed higher salt

tolerance by overexpression of OsNAC5. The role of NAC

gene in ABA signaling is reported by Liu et al. [56], the

Arabidopsis transgenic lines with AhNAC2 (Arachis hyp-

ogaea) showed hyper sensitivity to ABA in root growth,

seed germination, and stomatal closure compared to wild

type Arabidopsis. The AhNAC2 overexpressed lines

showed higher expression of downstream genes viz.

Rd29A, Rd29b, Rab18, AtMYB2, AtMYC2, ERD1, COR 47,

COR15a, KIN1, AREB1, CBF1, AMY1. The upregulation

of Rd29A, Rd29b can correlate the ABA signaling of Ah-

NAC2 since both the gene possess ABRE element in their

promoter region [56].

Myb

In higher plants, myb (myeloblastosis)-TF family repre-

sents a large number of genes. In Arabidopsis it is con-

sidered as the largest TF family and contains more than 163

genes [57]. Plant R2R3-MYB TFs play wide functional

roles, and involved in imparting stress tolerance against

various environmental cues in transgenics (Table 2). The

OsMYB3R-2I from rice employed enhanced tolerance to

salt, freezing and dehydration stresses and decreased sen-

sitivity to ABA in transgenic Arabidopsis [58]. In contrast,

Jung et al. [59] reported that the AtMYB44 transgenic were

highly sensitive to ABA and a rapid ABA induced stomatal

closure was seen during the stress treatment. Transgenic

plants had low rate of water loss and enhanced tolerance to

drought and salt stress compared to wild-type plants. The

AtMYB44 overexpressing lines revealed the low expression

of protein phosphatases 2C (PP2Cs) genes in microarray

analysis and northern blots analysis, whereas the mutant

lines showed the higher expression of PP2Cs and reduced

tolerance to salt and drought. This study showed that At-

MYB44 performs the abiotic stress function by suppressing

the negative regulator group of genes. Liao et al. [60] has

studied expression of large number of soybean Myb genes

under ABA, salt, drought, and/or cold stress. The overex-

pression of three Myb genes, viz., GmMYB76, GmMYB92,

or GmMYB177 in Arabidopsis showed higher seed germi-

nation rate under salt media [60]. Recently, Gao et al. [61]

reported that overexpression of apple Myb10 resulted in

osmotic stress tolerance. The transgenic apple plants

showed higher flavonoid content, thus manages higher

antioxidative ability to cope-up osmotic stress. Three dif-

ferent Myb TFs were isolated from wheat (Triticum aes-

tivum L)., TaMYB2A, TaMYB2B, and TaMYB2D [62].

TaMYB2A Arabidopsis transgenics showed enhanced tol-

erance to drought, salt, and freezing stresses and revealed

decreased rate of water loss, enhanced cell membrane

stability, improved photosynthetic potential, and reduced

osmotic potential. Another MYB gene TaPIMP1, was also

isolated from wheat: TaPIMP1 showed significantly higher

transcript level by a fungal pathogen Bipolaris sorokiniana

and by drought treatment. TaPIMP1 transgenic tobacco

lines showed tolerance to salinity along with pathogen and

drought. In these transgenic lines, the activities of phen-

ylalanine ammonia-lyase (PAL) and superoxide dismutase

(SOD) were significantly increased compared to wild-type

tobacco plants [63].The Solanum lycopersicum abscisic

acid-induced myb1 (SlAIM1) TF is reported to induced by

pathogens, plant hormones, salinity and oxidative stress,

suggesting a function in pathogen and abiotic stress

responses. The RNAi plant with silenced SlAIM1 showed

high susceptibility to fungus Botrytis cinerea, and high

sensitivity to salt and oxidative stress. These responses

correlate with reduced sensitivity to abscisic acid (ABA) in

the SlAIM1 RNAi, but increased sensitivity in the over-

expression plants, suggesting SlAIM1-mediated ABA

responses are required to integrate tomato responses to

biotic and abiotic stresses. The SIAIM1 also shows the

regulation of ion fluxes, the RNAi plants accumulated more

Na? compared to overexpressed lines [64].

Osmolytes

Osmolytes are organic metabolites of low molecular

weight known as compatible solutes and do not deter the

cellular functions. The osmolytes such as glycine betaine,

fructans, trehalose, mannitol, sorbitol, ononitol, and pinn-

itol play prominent role as osmoprotectants. Genes for

many osmolytes have been cloned and introduced into

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Table 2 Overexpression of transcription factors for salt and desiccation tolerance

Gene/source Transgenic plant Performance of transgenic plant References

HsDREB1A/Hordeum spontaneum Argentine bahiagrass Salt and dehydration tolerance [176]

PpDBF1/Physcomitrella patens Tobacco Salt, dehydration, and freezing tolerance [177]

DREB1A/Arabidopsis Arachis hypogaea Dehydration tolerance [178]

AIDFa/Triticum aestivum Arabidopsis Salt and dehydration tolerance [179]

DREB1A/Arabidopsis Festuca arundinacea Dehydration tolerance [180]

DgDREB1A/Dendranthema grandiflorum Arabidopsis Dehydration and freezing tolerance [181]

DREB1/Hordeum vulgare Arabidopsis Salt tolerance [182]

DREB2/populus Tobacco Salt tolerance [183]

DREB2A/Pennisetum glaucum Tobacco Salt and dehydration tolerance [184]

DREB2A/Salicornia brachiata E.coli (BL21DE3) Salt and dehydration tolerance [40]

DREB2B/rice Arabidopsis Dehydration and heat tolerance [185]

DREB2A/maize Arabidopsis Dehydration [186]

DREB2/Glycine max Arabidopsis Salt and dehydration tolerance [187]

DREB/cotton Wheat High salt drought and cold tolerance [188]

DREB1D/rice Arabidopsis Salt tolerance [189]

DREB2ACA/Arabidopsis Arabidopsis Drought tolerance [190]

OsDREB1F/rice Arabidopsis/Rice Salt and dehydration tolerance [191]

2 9 35S TaDREB2/wheat Barley and wheat Drought tolerance [192]

2 9 35S TaDREB3/wheat

pRAB17 TaDREB2/wheat

pRAB17 TaDREB3/wheat

LcDREB3a/Leymus chinensis Arabidopsis thaliana Salt and drought tolerance [193]

MtCBF4/Medicago truncatula Medicago truncatula Salt tolerance (enhanced root length in 10 mM NaCl) [194]

VrCBF1/grape Arabidopsis Drought tolerance [195]

VrCBF4/grape

ERF3/Glycine max Tobacco Salt Dehydration tolerance [196]

CpMYB10/Craterostigma plantagineum Arabidopsis Salt and desiccation tolerance [197]

OsMYB3R-2/rice Arabidopsis Salt, drought and freezing tolerance [58]

GmMYB76 or GmMYB177/Glycine max Arabidopsis Salt and freezing tolerance [60]

AtMYB44/Arabidopsis Arabidopsis Drought and salt tolerance [59]

Myb10/apple Arabidopsis Osmotic stress [61]

TaPIMP1(MYB)/wheat Tobacco Salt and drought tolerance [64]

AtMYB52/Arabidopsis Arabidopsis Drought tolerance [198]

TaMYB2A/wheat Arabidopsis Salt and drought tolerance [62]

SNAC2/Rice IRA109 Rice Zhonghua 11 Salt and cold stress tolerance [50]

NTL8(NAC)/Arabidopsis Arabidopsis GA mediated Salt signaling [199]

ONAC063/rice Arabidopsis Salt and Osmotic stress tolerance [51]

SNAC1/rice Rice Salt tolerance [49]

AhNAC2/groundnut Arabidopsis Salt and drought tolerance [56]

DgNAC1/Chrysanthemum Tobacco Salt tolerance [53]

GmNAC11/Glycine max Arabidopsis Salt tolerance [52]

GmNAC20/Glycine max

HvDhn4s:TaNAC69/wheat

Stress inducible

HvDhn8s:TaNAC69/wheat

Constitutive

Wheat In combined salt and drought by producing more biomass

Drought tolerance

[200]

OsNAC5/rice Arabidopsis

Rice

Salt and drought tolerance [54]

MYB myeloblastoma; NAC no apical meristem, ATAF1,2 and cup-shaped cotyledon; DBF DRE (drought responsive element) binding factor; DREBdrought responsive element binding protein

108 Mol Biotechnol (2013) 54:102–123

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plants. Generally, this resulted in higher accumulation of

osmoprotectants and enhanced salt and drought tolerance

(Table 3).

Trehalose is a non-reducing disaccharide and its syn-

thesis help in achieving dehydration tolerance to living

organism. It protects biomolecules by water replacement,

glass formation and chemical stability [65]. The genes for

trehalose synthesis have been cloned from bacteria, yeast,

and eukaryotic plants, and first time overexpressed in

tobacco enhancing drought tolerance by incorporation of E.

coli otsA gene or ScTPS1, trehalose-6-phosphate synthase

from yeast [66, 67]. Later, ScTPS1 gene was introduced

into Arabidopsis [68], alfalfa [69], rice, and tomato [70]

and the transgene imparted enhanced salt tolerance to the

plants. Garg et al. [71] introduced OtsA and OtsB (E. coli

trehalose-6-phosphate synthase) genes in rice and observed

increased tolerance to salt, drought, and cold in the trans-

genic plants. The trehalose-6-phosphate synthase gene

(OsTPS1) overexpressing rice lines showed improved tol-

erance to cold, high salinity and drought treatments without

any morphological changes. These lines also exhibited

higher expression of RAB16C, HSP70, and ELIP and Os-

TPP1 and OsTPP2 [72].

The E. coli mannitol-1-phosphate (mt1D) gene was

introduced into Arabidopsis [73], tobacco [74], and wheat

[75] plants. The transgenic Arabidopsis showed enhanced

seed germination on high salt (400 mM NaCl) medium

[73]. The wheat transgenic plants with mt1D showed less

reduction in biomass compared to wild-type plants under

the presence of salt and dehydration [75].

During stress treatment, the low molecular weight

metabolite proline gets accumulated in the cells. The bio-

synthesis of proline resulted in improved tolerance to salt

and drought stress in a number of crops [76, 77]. The proline

precursor, p5cs (D1-Pyroline-5-carboxylate synthase), has

been introduced into tobacco [78, 79], rice [80, 81], and

Arabidopsis [82, 83] and these transgenic plants showed

better tolerance to salinity stress.

Glycine betaine (betaine) is a non toxic cellular osmolyte

that raises intracellular osmolarity in response to different

stresses in lower organisms as well higher plants and sta-

bilize the biological macromolecules [84, 85]. Glycine

Table 3 Genetic transformation of osmolytes for salt and desiccation tolerance in plants

Gene/source Transgenic plant Performance of transgenic plant References

ScTPS1/yeast Tobacco Dehydration tolerance [67]

ScTPS1/yeast Arabidopsis Salt tolerance [68]

TPS1-TPS2/yeast Alfalfa Salt tolerance [69]

TPS1/yeast Tomato Salt tolerance [70]

OsTPS1/Oryza sativa Rice High salinity and drought tolerance [72]

OtsA and OtsB/E. coli Rice Salt, drought and cold tolerance [71]

mt1D/E. coli Arabidopsis High salt tolerance [73]

mt1D/E. coli Tobacco Salt and dehydration tolerance [74]

mt1D/E. coli Wheat Salt and dehydration tolerance [75]

MIPS/Potresia coarctata Tobacco Growth improvement at 300 mM NaCl [201]

mt1D and gutD/A. tumefaciens Loblolly pine/Pinus taeda Salt tolerance [202]

p5cs/Vigna aconitifolia Tobacco Salt tolerance [78]

p5cs/Vigna aconitifolia Rice Salt tolerance [80]

Antisense ProDH/Arabidopsis Arabidopsis Salt and freezing tolerance [82]

TaP5CR/Triticum aestivum Arabidopsis Salt tolerance [83]

p5cs/Vigna aconitifolia Rice Increased fresh weight at 200 mM NaCl [81]

p5cs/Vigna aconitifolia Wheat Salt tolerance [203]

p5cs/Vigna aconitifolia Tobacco Salt tolerance [79]

p5cs/Arabidopsis Solanum tuberosum Salt tolerance [204]

CodA/A. globiformis Arabidopsis, Rice, Brassica Salt tolerance [205– 208]

betA/E.coli B. oleracea Salt tolerance [89]

AhBADH/ Wheat Salt tolerance [209]

E. Coli betA and betB Tobacco Salt tolerance [85]

ADC/Oat Arabidopsis Drought tolerance [210]

TPS trehalose-6-phosphate synthase, p5cs D1-Pyroline-5-carboxylate synthase, codA Choline oxidase, BADH Betaine aldehyde dehydrogenase,

mt1D Mannitol-1-phosphate dehydrogenase, P5CR P5C reductase, GutD glucitol-6-phosphate dehydrogenase, MIPS L-myo-Inositol-1-phosphate

synthase

Mol Biotechnol (2013) 54:102–123 109

123

betaine is synthesized by two-step process in both pro-

karyotes and eukaryotes from choline with an intermediate

compound betaine aldehyde. In eukaryotes, choline gets

converted in betaine aldehyde with the help of choline

monooxygenase [86] in first step, which subsequently pro-

duce betaine in second step by betaine aldehyde dehydro-

genase (BADH) [87], whereas in bacteria the choline to

betaine aldehyde and finally to betaine is achieved by

choline dehydrogenase (CDH). However, several crop

plants are incapable of synthesizing betaine [85]. The E.

coli betA and betB genes [88] encoding for CDH and BADH

enzymes, respectively, were introduced in tobacco resulting

increased biomass and faster recovery from photoinhibition

under salt stress [85]. There are examples where only betA

gene has been overexpressed in B. oleracea [89] and maize

[90], which showed enhanced salt and drought tolerance,

respectively. In comparison to the CDH and CMO pathways

a direct choline oxidase pathway (COD) do exist which has

single step conversion of choline to glycinebetaine and also

does not require any cofactors for the catalysis [91]. A codA

(Choline oxidase) gene from Arthrobacter globiformis had

been introduced in many plants, resulting in enhanced

growth under salt stress which showed better germination

and growth in transgenic at 150 mM of NaCl (Table 3).

Antioxidative Enzymes

Salinity stress generates reactive oxygen species (ROS)

including singlet oxygen, superoxide anion radicals,

hydroxyl ions, and hydrogen peroxide [92–96] and consid-

ered as marker for stress activation in the plants [97]. ROS

serves as signaling molecules that regulates stress response to

maintain the growth and development of plants. In this

review the direct impact of oxidative stress compounds on

salt stress tolerance by genetic engineering approach is dis-

cussed. During salt stress available CO2 get reduced in the

leaf because of stomata closing, which further causes the

over-reduction of photosynthetic electron transport chain,

thereby leading to the generation of ROS [97]. Several

enzymes are involved in the detoxification of antioxidative

substances (AOS). Superoxide dismutase (SOD) converts

superoxide to H2O2, which is further scavenged by catalase.

Ascorbate peroxidase (APX) also reduces H2O2 and is

present in different isoforms in different plant organelles

[98]. Transgenic plants developed by overexpression of

several enzymes such as glutathione peroxidase (GPX),

SOD, APX, and glutathione reductases (GR) showed

improved stress tolerance (Table 4). Appearance of APX

isoenzymes in response to environmental stresses, such as

salinity and drought, has been reported in several plant spe-

cies and their overexpression led to salt and drought tolerance

in tobacco and Arabidopsis [99–101]. A number of SOD

isoforms are present in different plants as Cu/Zn-SOD iso-

forms is found in the chloroplast and in the cytosol, whereas a

Mn-containing enzyme is located in the mitochondria

showed high photosynthetic rate under increased salt stress in

tobacco transgenic plants [102]. A cytosolic Cu/ZnSOD gene

from the mangrove species, Avicennia marina enhanced salt

tolerance in the indica rice variety [103]. Similarly,

Table 4 Genetic transformation with antioxidative enzyme genes for salt and desiccation tolerance

Gene/source Transgenic plant Response of transgenic plant References

Chl-APX5/Arabidopsis Tobacco Salt and water stress tolerance [99]

APX/pea Tomato Salt tolerance [211]

APXa and APXb/rice Arabidopsis Salt tolerance [100]

APX/tomato Tobacco Salt and osmotic tolerance [98]

GST and GPX/tobacco Tobacco Salt tolerance [107]

GST/Suaeda salsa Arabidopsis Salt tolerance [212]

MnSOD/Arabidosis Arabidopsis Salt and Cold tolerance [104]

MnSOD/pea Rice Drought tolerance [213]

Cu/ZnSOD/Pea, APX and DHAR/human Tobacco Salt and oxidative tolerance [105]

Cu/Zn-SOD/A. marina Rice Salt, drought, and oxidative tolerance [103]

SOD/tomato Pepper Drought tolerance [214]

Cu/Zn-SOD/pea and APX Tobacco Salt and osmotic stress [106]

DHAR1/rice Arabidopsis Salt tolerance [215]

MDAR/Arabidopsis Tobacco Salt, ozone, and PEG [216]

AmMDAR/A. marina Tobacco Salt tolerance [217]

katE/E. coli Tobacco Resistance of chloroplast translation machinery to salt stress [218]

katE/E. coli Rice Salt tolerance [219]

APX cytosolic ascorbate peroxidase, DHAR dehydroascorbate reductase, MDHAR monoDHAR, SOD superoxide dismutase

110 Mol Biotechnol (2013) 54:102–123

123

overexpression of MnSOD showed enhanced salt tolerance in

Arabidopsis [104]. Many studies have been performed to

develop abiotic stress tolerant plants by engineering one gene

but recently interest has changed to incorporate more than

one gene for empowering better tolerance. However, on this

aspect only a few reports have appeared [105], transforma-

tion of tobacco plants by pyramiding more than one gene for

anti-oxidative enzyme like Cu/ZnSOD (C), APX (A), and

DHAR (D) into chloroplasts and demonstrated that such

plants were more salt and oxidative stress tolerant compared

to those expressing single or double gene transfer. The

overexpression of ‘CAD’ showed approximately 1.6–2.1

times higher dehydroascorbate reductase (DHAR) activity,

higher ratios of reduced ascorbate to dehydroascorbate

(DHA), and oxidized glutathione (GSSG) to reduced gluta-

thione (GSH) compared to ‘CA’ plants. The transgenic

seedlings with ‘CAD’ showed enhanced tolerance to NaCl,

compared to ‘CA’ plants. The ‘CA’ plants exhibited better

seed longevity compared to non-transgenic plants [106].

In addition to other reported antioxidative genes, GST

gene from a rice variety ‘Pusa Basmati-1’ showed better

growth under salt and cold stresses by scavenging ROS and

preventing membrane damage in transgenic tobacco plants

[107, 108]. Antioxidant compounds are proved as good

candidates for reducing the cellular toxicity and help plants

in maintaining good health in adverse environmental con-

ditions. The manipulation of antioxidant genes in plants

seems a good approach for keeping ROS at basal level.

Although, several reports have mentioned that (Table 4)

higher expression of single antioxidant gene control salt-

induced oxidative stress efficiently, but it is plausible to

incorporate more than one genes for balanced ratio of

protective enzymes and other metabolites.

Polyamines

The polyamines play essential roles in many fundamental

cellular processes, gene expression, protein synthesis, cell

division, cell differentiation, growth, development, and cell

death [109]. They are also involved in various abiotic and

biotic plant stress responses. The role of polyamines is

amply described in recent reviews [110, 111]. The heter-

ologous overexpression of ornithine decarboxylase (ODC),

arginine decarboxylase (ADC), S-adenosyl methionine

decarboxylase (SAMDC), and spermidine synthase (SPDS)

in rice, tobacco, and tomato has shown tolerance against

stress conditions (Table 5). In carrot cell lines the over-

expression of mouse ODC, which converts ornithine to

putrescine, exhibited high level of salt stress tolerance

[112]. Kumria and Rajam [113] also showed that mouse

ODC when introduced into tobacco exhibited enhanced

production of constitutively expressed putrescine, confer-

ring salt tolerance to the plants. A cDNA for ADC from oat,

expressed in rice under an ABA-inducible promoter,

showed higher biomass accumulation in salinity stress as

compared to the control plants [114]. The overexpression

of SAMDC1 in Arabidopsis led to elevated spermidine

levels and enhanced tolerance to various abiotic stress

conditions. Introduction of human SAMDC into tobacco led

to overexpression of putrescine and spermidine resulting in

enhanced salt and osmotic tolerance [115]. Different

examples confirmed that enhanced expression of poly-

amines control the salt and drought tolerance in plants.

However, a gap remains in understanding the mechanism

of polyamines involved in abiotic stress tolerance.

Transporter Genes

Plants apply both ionic and osmotic homeostasis to re-

establish themselves in saline environmental conditions.

Plants employ various strategies for maintaining low Na?

in the cell either by the active exclusion by the plasma

membrane Na?/H? antiporter AtSOS1 [116, 117], or by

sequestration of excess sodium into the vacuoles via vac-

uolar Na?/H? antiporters. Transporter proteins are impor-

tant candidates for genetic engineering to develop salt

Table 5 Overexpression of polyamine gene for salt and desiccation tolerance

Gene/source Transgenic plant Performance of transgenic plant References

ADC/oat Rice Salt tolerance [114]

ADC/Datura Rice Drought tolerance [220]

ADC2/Arabidopsis Arabidopsis Drought tolerance [221]

ODC/mouse Tobacco Salt tolerance [114]

SAMDC/Tritodeum Rice Salt tolerance [222]

SAMDC/human Tobacco Salt and osmotic tolerance [115]

SPDS/Cucurbita ficifolia Arabidopsis Salinity, hyperosmosis tolerance [223]

SPDS/apple Pear Salt and drought tolerance [224]

ADC arginine decarboxylase, ODC ornithine decarboxylase, SAMDC S-Adenosyl methionine decaroboxylase, SPDS spermidine synthase

Mol Biotechnol (2013) 54:102–123 111

123

Table 6 Overexpression of transporter genes for salt and desiccation tolerance

Gene/source Transgenic plant Performance of

transgenic plant

References

NHX1/Arabidopsis cotton, Arabidopsis, tomato,

B. napus Festuca, Fagopyrum,sugar beet, kiwi fruit, wheat, maize

Salt tolerance [44, 119, 163, 225–231]

NHX1/Atriplex gmelini Rice Salt tolerance [232]

NHX1/wild type rice Rice Salt tolerance [233]

NHX1/Gossipium Tobacco Salt tolerance [234]

NHX1/Hordeum Tobacco Salt tolerance [235]

NHX1/Pennisetum B. juncea, rice Salt tolerance [236, 237]

NHX1/Agropyron elongatum Arabidopsis/Festuca Salt tolerance [238]

NHX1/Reed Yeast Salt tolerance [56]

NHX1/Aleuropus littoralis Tobacco Salt tolerance [239]

NHX1/Salicornia brachiata Tobacco Salt tolerance [124]

NHX1/Salsola soda Alfalfa Salt tolerance [240]

NHX1/Malus Apple Salt tolerance [241]

NHX1/Thellungiella halophilla Arabidopsis Salt tolerance [242]

HcNHX1/Halostachys caspica Arabidopsis Salt tolerance [243]

AtNHX1/Arabidopsis Arachis hypogaea Salt tolerance [244]

AmNHX2/Ammopiptanthus mongolicus Arabidopsis Salt and drought tolerance [245]

NHX2/Hordeum Potato Salt tolerance [246]

HAL1/yeast Cucumis melo, tomato Salt tolerance [125, 126]

nhaA/E. coli Rice Salt tolerance [130]

SOD2/yeast Arabidopsis, rice Salt tolerance [129, 247]

SOS1/Arabidopsis Arabidopsis Salt tolerance [117]

SOS1/rice Yeast Salt tolerance [248]

SOS1/Populus euphratica E. coli Salt tolerance [249]

SOS1-RNAi/Thellungiella halophilla Thellungiella halophilla Salt tolerance [131]

SOS1

SOS1-RNAi/Solanum lycopersicon

Yeast

Tomato

[250]

NHX1,SOS1, SOS3, SOS2 ? SOS3, NHX1 ?

SOS3, SOS1 ? SOS2 ? SOS3/ArabidopsisArabidopsis Salt tolerance [132]

Avp1(H?-PPases)/Arabidopsis Arabidopsis Salt tolerance [135]

H?-PPase/Rhodospirillum rubrum Tobacco Salt tolerance [251]

TsVP(H?-PPase)/Thellungiella halophila Tobacco, maize, cotton Salt tolerance [252–254]

Avp1(H?-PPases)/Arabidopsis Alfalfa Salt tolerance [255]

Avp1/Arabidopsis ? NHX1/Saueda salsa Rice Salt tolerance better than

SsNHX1 alone

[256]

SsVP1/Saueda salsa Arabidopsis Salt and drought tolerance [257]

H?-PPase TVP1 and TNHX1/wheat Arabiodopsis Salt and water deprivation

tolerance

[133]

Avp1/Arabiodopsis Cotton, Creeping bent grass Salt and drought tolerance [258, 259]

TsVP/Thellungiella halophila Cotton Salt tolerance [260]

ATPase pENA1/Physcomitrella patens Rice Salt tolerance [261]

V-ATPase c subunit gene ThVHAc1/

Tamarix hispidaSaccharomyces cerevisiae Salt and drought tolerance [262]

NHX-1 vacuolar Na?/H? antiporter, SOS1 plasma membrane Na?/H? antiporter, TsVP H?-pyrophosphatase, HKT2 high efficiency potassium

transport

112 Mol Biotechnol (2013) 54:102–123

123

tolerant plants. Of late, several efforts have been made to

develop salt tolerant plants by upregulation of transporter

genes as compared to other classes of genes (Table 6). A

gene, homologous to animal plasma membrane Na?/H?

antiporters of the NHE family and the yeast ScNHX1 was

first identified from Arabidopsis genome and termed At-

NHX1 [118]. Overexpression of Arabidopsis AtNHX1

conferred enhanced salt tolerance in Arabidopsis [119].

Na?/H? antiporters have been isolated from several gly-

cophytes and halophytes, i.e., Mesembryanthemum crys-

tallinum [120], Atriplex gmelini [121], Saudea salsa [122],

Beta vulgaris [123], and Salicornia brachiata [124]. The

yeast HAL1 gene showed a certain level of salt tolerance

when expressed in transgenic melon [125] and tomato

[126] plants and retained more K? than the control plants

under salt stress. There are more than 26 reports on

acquiring salt tolerance induced by overexpression of

NHX. These include five halophytic plants (Table 6).

Salt overly sensitive (SOS) pathway was found to be

involved in Na? exclusion. SOS1 is a plasma membrane

Na?/H? antiporter that excludes Na? by taking H? into the

cytoplasm. The SOS pathway is regulated by Ca2?

dependent protein kinase signaling [14]. SOS pathway

involves SOS1, SOS2 and SOS3. Ca2? signaling is per-

ceived by SOS3, a calcium binding protein. SOS3 activates

SOS2, a protein kinase that activates SOS1 by its phos-

phorylation. Recently, SOS4 and SOS5 have also been

characterized by Shi et al. [127]. SOS pathway also regu-

lates vacuolar Na?/H? antiporter exchange activity and

Na? compartmentalization [128]. SOS1 gene from Ara-

bidopsis was ectopically expressed first time in Arabidopsis

plant showed reduced Na? accumulation in the presence of

salt [117]. Similar results were obtained when the plasma

membrane Na?/H? antiporters, SOD2 (Sodium 2) from

Schizosaccharomyces pombe and nhaA from Escherichia

coli, were overexpressed in Arabidopsis [129] and rice

[130], respectively. The SOS1 from Thellungiella salsugi-

nea suppressed the salt sensitive phenotype when expres-

sed in the yeast cells. This gene showed high salt stress

tolerance in the Arabidopsis transgenic plants, when the

SOS1 gene was suppressed by ThSOS1-RNAi in Thel-

lungiella salsuginea, the plants showed high salt sensitivity

compared to wild-type plants [131].

Instead of transforming single transporter genes, some

researchers have tried manipulation of a combination of two

or more transporters in plants. Yang et al. [132] tested

overexpression of multiple genes to improve salt tolerance

in Arabidopsis. They produced six different transgenic

Arabidopsis plants overexpressing AtNHX1, SOS1, and

SOS3 alone or in different combinations (AtNHX1 ? SOS3,

SOS2 ? SOS3, SOS1 ? SOS2 ? SOS3). Surprisingly, the

AtNHX1 alone did not show significant salt tolerance. In

220 mM NaCl treatment for 3 days, less than 20% of the

control and transgenic plants overexpressing only AtNHX1

survived, but over 80% of the transgenic plants over-

expressing SOS1, SOS3, SOS2 ? SOS3, AtNHX1 ? SOS3,

or SOS1 ? SOS2 ? SOS3 survived [132]. Brini et al. [133]

reported that overexpression of wheat TVP1 (Tonoplast H?-

PPase) and NHX1 in Arabidopsis conferred better growth in

the presence of 200 mM NaCl and also under a water-

deprivation regime, while wild-type plants exhibited chlo-

rosis and growth inhibition. The proton pumps present at the

cellular membrane work as driving force for nutrient uptake

[134]. Three distinct proton pumps are responsible for the

generation of the proton electrochemical gradients: (1) the

plasma membrane H-ATPase pump (PM H-ATPase), (2)

vacuolar type H?ATPase (V-ATPase), and (3) the vacuolar

H-pumping pyrophosphatase (H-PPase). The PM H-ATP-

ase extrudes H? from the cell and thus generates a proton

motive force while V-ATPase and H-PPase acidify the

vacuolar lumen and other endomembrane compartments

[12]. Genetic evidence of physiological role of PM

H-ATPase and V-ATPase is very scarce, whereas, a few

studies have been carried out on H-PPase showing its

involvement in salt tolerance. The pioneer work was carried

out by Gaxiola et al. [135], overexpression of Avp1 (H?-

PPase) showed enhanced salt tolerance in transgenic Ara-

bidopsis plants. Later this gene was isolated from Thel-

lungiella halophila and engineered in tobacco, maize, and

cotton showed enhanced salt tolerance (Table 6). As evi-

dent from the literature (Table 6) a large interest have been

shown for overexpressing the transporter genes in model as

well as crop plants. In future, these transgenic plants should

be tested in the field to test the efficacy in the adverse

environment conditions.

Glyoxalase Pathway

Glyoxalase pathway has emerged as a prospective can-

didate for the genetic engineering of salt tolerance. The

glyoxalase pathway has shown its role in rapidly dividing

plant cells [136, 137] and in stress tolerance [138–140].

This pathway involves two enzymes, glyoxalase I and

glyoxalase II, which convert methylglyoxal to lactic acid

in two-step reactions [7]. Methylglyoxal is a potent

cytotoxic compound, and is the primary substrate for

glyoxalase I. Expression of the GlyI gene from Brassica

juncea showed salt tolerance in transgenic tobacco lines

[139]. Singla-Pareek et al. [141] achieved better salt stress

tolerance in tobacco by overexpression of both GlyI and

GlyII together. Further, Singla-Pareek et al. [142] trans-

formed only GlyII in rice plants, which showed higher

tolerance to NaCl.

Mol Biotechnol (2013) 54:102–123 113

123

Role of Small RNA in Abiotic Stress Tolerance

Recently, a newer approach to understand the mechanism

of abiotic stress in plant system through miRNA/siRNA

approach is getting attention. The plant response to the

stress relies on the correct regulation of gene expression,

which depends on the number of mechanisms. The post-

transcription events play a crucial role in regulating gene

expression at a right time [143]. MicroRNA and short

interfering RNA are small (approx. 21 nt) non-coding

RNA, involved in regulating gene expression by mRNA

degradation, translational repression, and chromatin mod-

ification [144, 145]. The small RNA regulate gene

expression in stressed plants either by overexpression of

small RNA which resulted in downregulation of negative

regulators, or reduced biogenesis of small RNA which

causes downregulation of positive regulators and accumu-

lation of beneficial gene products [143].

Recently, a number of reports have been published

elucidating the role of microRNAs in regulating gene

expression under various environmental conditions [146–

151]. Some good reviews on role of microRNA in stress

response elucidating their roles have also been published

[143, 152, 153]. Several miRNAs have been isolated dur-

ing salt treatment from Arabidodpsis [144], P. trichocarpa

[154], and rice [155] plants. A detailed mechanism of

miR398 has been studied by Sunkar et al. [149], where

mRNA abundance of Cu/Zn SOD1 (CSD1, cytosolic) and

Cu/Zn SOD2 (CSD2, chloroplastic) was observed inversely

correlated by the abundance of miR398 under salt stress.

There are very limited studies on the overexpression of

miRNA for abiotic stress tolerance. Atmir398 from Ara-

bidopsis was overexpressed in tobacco plants, caused

reduced seedling and root growth by down regulating

NtTIR1 expression and causing auxin insensitivity in

transgenic tobacco plants. These transgenic plants showed

enhanced resistance to salt stress by suppressing auxin

signal via degradation of NtTIR1 mRNA [156]. The rice

osa-MIR396c showed a dramatic transcript change under

salt and alkali stress conditions in Oryza sativa [157]. The

osa-MIR396c, osa-MIR393 transgenic rice, and Arabidop-

sis lines showed that several TFs related to growth, devel-

opment, and stress tolerant genes are targeted by these

microRNA and eventually showed reduced salt and alkali

stress tolerance compared to that of wild-type plants [157,

158]. Xia et al. [159] observed that OsmiR393 has same

results in transgenic rice as reported by Gao et al. [158] in

response to salt. The transgenic lines also showed two new

functions as increased tilling and early flowering. However,

the report of Gao et al. [157] is in contradiction to AtmiR396

overexpression in Arabidopsis [160], which conferred the

tolerance to drought and other stresses. The overexpression

of Arabidopsis miR395c or miR395e retarded and accel-

erated the seed germination of Arabidopsis under high salt

or dehydration stress conditions, respectively. The overex-

pression of miR395c and miR395e, did not show the same

cleavage of mRNA targets, APS1, APS3, APS4, and

SULTR2;1, in both the overexpressing plants. Wang et al.

[161] reported suppression of an Arabidopsis bacterial-type

PEPC gene, Atppc4, by artificial microRNA (amiRNA).

Atppc4-amiRNA transgenic plants showed decreased

accumulation of Atppc4 transcripts, whereas other three

plant-type PEPC genes, Atppc1, Atppc2, and Atppc3 were

significantly upregulated and these transgenic plants

showed improved tolerance to salt stress.

These results demonstrated that a given miRNA family

containing a single nucleotide difference can guide the

cleavage of various mRNA targets, thereby acting as a

positive or negative regulator of seed germination under

stress [162]. These studies highlight that microRNA play

an important role in targeting various important genes for

managing stress tolerance in plants. Recently miRNAs is

getting increased attention and in near future a vast

knowledge on the mechanism of miRNA for abiotic stress

will be generated.

Conclusion

Genetic engineering towards developing salt and drought

tolerant crops is challenging and forefront area of research

for future crop improvement programs. Conventional

breeding has made considerable success in twentieth cen-

tury to improve crop yield and quality but limited work was

carried out for developing abiotic stress tolerant crop plants

with only few successful reports [9]. This is probably due

to the low magnitude of genetically based variation in the

plants for salinity tolerance. For achieving the salinity

tolerance, the genetic material from the distant wild rela-

tives or halophytes can be used for transfer the salt tolerant

genes into the sensitive plants through conventional

breeding but again the incompatibility of the reproduction

hinders in achieving the goal. With the advent of plant

molecular biology and understanding of the stress signal

transduction pathways, it is now possible to generate plants

with least damaging effect on environmental conditions

and concurrently promising an increase in productivity. So

far, many stress-related genes have been isolated and

characterized in the model plants like tobacco and Ara-

bidopsis. With the progress of whole genome sequencing

of different plant species, it seems now easier to identify

unique stress responsive genes. During the last decade,

transgenic plants with stress tolerant genes have been

generated using various genes from diverse pathways

114 Mol Biotechnol (2013) 54:102–123

123

(Tables 1–6). However, abiotic stress tolerant plant varie-

ties are yet to be released for the benefit of the farmers. The

sincere effort in this area is needed, in most cases trans-

genic plant performance are being evaluated under con-

trolled growth environments. Therefore, a gap remains

between the success in the laboratory and the application of

these techniques to develop the crops in the field. The

performance of the transgenic plants for salt tolerance

should be carried out in the laboratory by mimicking dif-

ferent stress together as in the field. Therefore, this area is a

serious concern and much attention needs to be focussed

towards the plants response to a combination of stresses.

Attempts made by Xue et al. [163] and Waterer et al. [164]

for evaluating the transgenic wheat and potato plants,

respectively, in the field to asses abiotic stress tolerance are

paragons. The AtNHX1 transgenic wheat lines produced

higher grain yields and heavier and larger grains in the field

of saline soils [163]. The potato transgenic lines showed

the higher yield in drought condition [164].

The basic genetic studies have shown that the stress

tolerant traits are multigenic in nature. Therefore, it is

significant to transfer multiple genes for better control of

stress tolerance traits. However, as evident from the tables,

there are very few studies, in which more than one gene has

been introduced [106, 132] into the same plant to confer

better stress tolerance.

A meticulous fine-tuning of the expression of the known

candidate genes for stress tolerance in specific temporal

and spatial patterns is also one of the essential parameters

for avoiding negative effects in plant growth and devel-

opment. The usage of specific TFs controlled by stress

inducible promoters could be a good choice for developing

diverse stress tolerance without compromising plant

energy. The genetic engineering of stress tolerance can

develop future crops to survive better in adverse environ-

mental conditions and lead to agricultural benefits.

Acknowledgments The financial support received from CSIR and

DST New Delhi is gratefully acknowledged. Kapil Gupta acknowl-

edges the award of CSIR-SRF.

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